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Microbes & Immunity Dynamics between phage, bacteria, and mammalian cells

2.2. Phage counting In the three-component system, the viability of

The number of viable phages in stock suspensions and BEAS-2B cells after various treatments was also assessed
samples after treatment was determined using the Miles– using an MTT assay. Briefly, the supernatant of each well
Misra surface droplet technique. Serial 10-fold dilutions was removed and washed twice with PBS. The remaining
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were performed by adding 20 μL samples to 180 μL PBS. bacteria were killed by incubating 3× minimum inhibitory
A volume of 200 μL host bacteria (OD ~1) was mixed with concentrations (MIC) of colistin solution (6 μg/mL),
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600
5 mL molten soft agar (0.7% agar, 48°C) and overlayed onto determined using the broth microdilution method, for
a solidified nutrient agar made of 1.5% agar and nutrient 3 h to minimize its impacts on the MTT assay. Thereafter,
broth. A volume of 10 μL diluted phage samples was dropped the spent medium was replaced with 190 μL DMEM and
onto the agar lawn in triplicate, air-dried, and incubated at 10 μL MTT solution (5 mg/mL, Sigma Aldrich, USA) and
37°C overnight. Diluted samples, yielding 3 – 30 plaques, incubated in a 5% CO humidified incubator at 37°C for 4 h.
2
were used for phage viability calculation using the equation: After removing the medium, DMSO was added to dissolve
the formazan crystals for optical density measurement
Phage titer (PFU/mL) = Average plaque counts × Dilution (OD ) using a microplate reader (Multiskan FC, Thermo
570
factor × 100 (I) Scientific, USA). The control group treated with PBS was
defined as 100% survival. The survival ratio of phage-
2.3. Bacterial viability assessment
treated groups was calculated according to the ratio of
The bacteria viability in PBS and phage-treated samples was absorbance values between the phage-treated and PBS-
determined through plate counting. Briefly, 20 μL samples treated groups.
were pipetted out for 10-fold dilution with PBS. A volume
The experiment was performed in biological replicates
of 10 μL diluted phage samples was dropped onto the (n = 6) and repeated in two independent experiments. After
nutrient agar plate in triplicate, air-dried, and incubated the investigation with AB406 phages, the experimental
at 37°C overnight for bacterial plating. Bacterial colonies conditions were set for simultaneous co-incubation
(3‒30 valid) visible to the naked eye were counted using a
manual counter. The corresponding colony-forming unit (Condition ii) at a phage-to-bacteria MOI of 100:1 to
examine the effects of other phage types (AB2 and AB9) in
(CFU/mL) was calculated similarly to the phage counting the co-culture system.
described above.
2.5. Adsorption assays
2.4. In vitro antibacterial efficiency efficiency in a
three-component coculture system Adsorption of phages and bacteria onto the epithelial
cell surface was evaluated to elucidate their physical
The AB406 phage was first used to study the antibacterial interactions in the co-culture system.
effects of various incubation conditions with the human
lung epithelial cell line BEAS-2B (three-component). 2.5.1. Phage-epithelial cell adsorption assay
BEAS-2B cells were seeded in a 96-well plate at 3 × 10 cells
4
per well in 100 μL of DMEM culture medium. After BEAS-2B cells were seeded in a 96-well plate at 3 ×
4
overnight incubation, the spent medium was removed and 10 cells per well in 100 μL DMEM culture medium. After
three incubation conditions were examined (Figure 1A): overnight incubation, the spent medium was removed, and
9
(i) 90 μL bacteria (~10 CFU/mL) pre-incubated with the cells were further incubated with 100 μL phage (~10
7
BEAS-2B cells for 4 h, followed by co-incubation with 10 PFU/mL) suspended in DMEM in the absence or presence
7
μL AB406 phage for 24 h; (ii) 90 μL bacteria and 10 μL of bacteria (~10 CFU/mL). After 24-h incubation at 37°C
AB406 phage co-incubated simultaneously with BEAS-2B and 5% CO , the wells were washed 3 times with PBS
2
cells for 28 h; and (iii) 10 μL AB406 phage pre-incubated to remove any unadsorbed phages. Thereafter, 100 μL
with BEAS-2B cells for 4 h, followed by co-incubation PBS containing 2% Triton X-100 was added to the lysate
with 90 μL bacteria for 24 h at 37°C and 5% CO . Different cells, following vigorous pipetting. A 20 μL sample was
2
phage-to-bacteria ratios (multiple of infection [MOI]: 1:1, withdrawn from each well for plaque assay to quantify
10:1, 100:1, and 1000:1) were studied. After the incubation the adhered/internalized phages. The experiment was
period, an aliquot of the bacterial culture (20 μL) was taken performed in biological replicates (n = 3) and repeated two
after vigorous pipetting for bacterial and phage counting. independent times.
The antibacterial efficiency of AB406 phage against The adherence of phages onto the BEAS-2B cells
MDR-AB2 bacteria in the absence of BEAS-2B cells was was also visualized with fluorescence staining assay
evaluated along with the three-component system for direct using fluorescein isothiocyanate (FITC)-labeled phages
comparison. according to reported procedures. Briefly, the phage
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Volume 1 Issue 1 (2024) 83 doi: 10.36922/mi.3141